Project Summary/Abstract The broad objective of this proposal is to understand the molecular mechanism of homologous recombination in humans, and to understand how the consequences of defects in recombinational DNA repair result in chromosomal instability and predisposition to cancers. Understanding the functions of key proteins in homologous recombination, many of which are tumor suppressors, will provide insight into how mutations in these proteins can predispose individuals to cancer. We plan to elucidate the biochemical roles and examine the mechanism of BRCA1, BLM, EXO1, PALB2, WRN, and the RAD51 paralogs functions, as well as the consequences of the interactions between these proteins, to provide insight into their role in recombinational DNA repair. We plan to reconstitute the initial steps of human recombinational DNA repair, and thereby understand the biochemical functions of these proteins. In addition, we will use single-molecule imaging to reveal the molecular mechanisms by which these proteins act.